Bone Marrow Adipose Portion Isolation Device and Methods

ABSTRACT

The embodiments disclosed herein generally relate to systems, devices and methods for the fractionation, isolation, extraction and processing of the adipose supernatant layer of a bone marrow aspirate. In particular, the various embodiments relate to systems devices and methods of obtaining, utilizing and processing the adipose supernatant layer of a bone marrow aspirate as a source of mesenchymal stem cells.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.14/778,530, filed on Sep. 18, 2015, entitled “Bone Marrow AdiposePortion Isolation Device and Methods”, which is a 35 U.S.C. § 371national phase application of PCT/US14/49992 (WO 2015/021189), filed onAug. 6, 2014, entitled “Bone Marrow Adipose Portion Isolation Device andMethods”, which application claims the benefit of U.S. ProvisionalApplication Ser. No. 61/862,837, filed Aug. 6, 2013, which isincorporated herein by reference in its entirety.

BACKGROUND

Adult mesenchymal stem cells (MSCs) are capable of robust tissue repair.MSCs can be isolated from many autologous tissue sources with the twomost common sources being adipose tissue and bone marrow. Adipose stemcell harvesting is performed through liposuction of subcutaneous fattissue which is then usually processed with a chemical digestiontechnique. Bone marrow aspirate is a red liquid when first obtained fromthe patient through a trocar inserted through the bony cortex. Theaspirate fluid is then typically processed with centrifugation toseparate out various marrow fractions (referred to herein as fractionsor layers). The buffy coat is a middle fraction of centrifuged marrow,positioned below a serum component and above a red blood cell component.The buffy coat is rich in nucleated cells, progenitor cells, and stemcells.

Until recently, only the buffy coat of a fractionated bone marrowaspirate was known to include useful quantities of MSCs. Accordingly,typical bone marrow fractionation and concentration systems designed toisolate a therapeutically significant quantity of MSCs have focused onisolating and processing the buffy coat through various means.Conventional methods and devices do not provide for the isolation orsubsequent processing of other marrow fractions that are now known tocontain MSCs, in particular a marrow adipose layer supernatant which ispositioned above the serum layer when bone marrow aspirate isfractionated. Therefore, according to conventional techniques, themarrow adipose supernatant layer is discarded as waste.

Although the adipose layer supernatant of fractionated bone marrowaspirate is now known to include MSCs, no techniques are known forefficiently collecting the adipose layer supernatant and processing sameto maximize a useable MSC yield.

The embodiments disclosed herein are directed toward overcoming one ormore of the problems discussed above.

SUMMARY OF THE EMBODIMENTS

The embodiments disclosed herein generally relate to systems, devicesand methods for the fractionation, isolation, extraction and processingof the adipose supernatant layer of a bone marrow aspirate. Inparticular, the various embodiments relate to systems, devices andmethods of obtaining, utilizing and processing the adipose supernatantlayer of a bone marrow aspirate as a source of mesenchymal stem cells.

One embodiment is a method of processing bone marrow aspirate utilizinga device having a first chamber, a second chamber in fluid communicationwith the first chamber, and a mechanical emulsification system in fluidcommunication with the second chamber. The method includes fractionatingbone marrow aspirate within the first chamber of the device into layersincluding an adipose layer supernatant. The adipose layer supernatant iscollected from the processed bone marrow aspirate in the second chamber.In addition, the adipose layer supernatant is emulsified in theemulsification system.

Method embodiments may further include processing the adipose layersupernatant to collect mesenchymal stem cells. In certain instances, asecondary substance will be added to the bone marrow aspirate. Secondarysubstances may be, for example, a biologically inert fluid, CaCl₂,thrombin, a clotting agent or a polymerization agent. Alternatively, adigestion agent may be added to the collected adipose layer supernatant.A digestion agent may be added separately or, in conjunction withanother secondary substance. Representative digestion agents includecollagenase or lecithin.

In certain embodiments, the method further includes applying at leastone of sonic energy or vibration to the adipose layer supernatant.

The second chamber may be a chamber of any type, including but notlimited to, a syringe, pipette or tube in fluid communication with theadipose layer supernatant. The first chamber may also be a chamber ofany type, and may include supplemental structures including but notlimited to, a cap having a fluid access port providing for the secondchamber to be placed into fluid communication with the adipose layersupernatant; a plunger providing for the expulsion of a selected portionof the fractionated bone marrow aspirate from the first chamber; a diskshaped volume which provides for the collection of an adipose layersupernatant fraction at a central region of the disk shaped volume uponthe rotation of the first chamber around a central axis; a portion ofrestricted diameter positioned to correspond with the location of anadipose layer supernatant fraction upon fractionation of bone marrowaspirate placed within the first chamber; a floating disk having adensity selected to cause the disk to float substantially between aserum layer and an adipose layer supernatant fraction upon fractionationof the bone marrow aspirate; a porous lipophilic membrane providing forthe separation of an adipose layer supernatant fraction uponfractionation of the bone marrow aspirate; or one or more ports in fluidcommunication with an adipose layer supernatant fraction uponfractionation of the bone marrow aspirate.

The mechanical emulsification system may, in some embodiments, include afirst emulsification chamber and a second emulsification chamber influid communication with each other through an aperture sized to provideemulsification upon passage of the adipose layer supernatant between thefirst and second emulsification chambers. Alternatively, the mechanicalemulsification system may include a first emulsification chamber and asecond emulsification chamber in fluid communication with each otherthrough an emulsification screen providing for the emulsification of theadipose layer supernatant upon passage of adipose layer supernatantbetween the first and second emulsification chambers. Alternatively, themechanical emulsification system may include an emulsification screenmovable with respect to the adipose layer and providing for theemulsification of the adipose layer supernatant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing selected layers present infractionated bone marrow aspirate.

FIGS. 2A, 2B, and 2C are schematic diagrams of one embodiment of deviceuseful for the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIGS. 3A, 3B, and 3C are schematic diagrams of another embodiment ofdevice useful for the collection, isolation and/or processing of theadipose supernatant layer of a bone marrow aspirate.

FIG. 4 is a schematic diagram of another embodiment of device useful forthe collection, isolation and/or processing of the adipose supernatantlayer of a bone marrow aspirate.

FIG. 5 is a schematic diagram of another embodiment of device useful forthe collection, isolation and/or processing of the adipose supernatantlayer of a bone marrow aspirate.

FIGS. 6A, 6B, and 6C are schematic diagrams of another embodiment ofdevice useful for the collection, isolation and/or processing of theadipose supernatant layer of a bone marrow aspirate.

FIGS. 7A and 7B are schematic diagrams of another embodiment of deviceuseful for the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIGS. 8A and 8B are schematic diagrams of another embodiment of deviceuseful for the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIGS. 9A and 9B are schematic diagrams of two alternative embodiments ofdevice useful for the collection, isolation and/or processing of theadipose supernatant layer of a bone marrow aspirate.

FIG. 10 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 11 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 12 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 13 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 14 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 15 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 16 is a schematic diagram of another embodiment of device usefulfor the collection, isolation and/or processing of the adiposesupernatant layer of a bone marrow aspirate.

FIG. 17A, 17B, and 17C are schematic diagrams of another embodiment ofdevice useful for the collection, isolation and/or processing of theadipose supernatant layer of a bone marrow aspirate.

FIG. 18 is a graphic representation of data showing the manual count ofmononucleated cells per 10 cc of bone marrow aspirate for all cellsobtained from the buffy coat and the adipose supernatant layer.

FIG. 19 is a graphic representation of flow cytometry data showing thepercentage of MSCs to total cell count for MCSs obtained from the buffycoat and the adipose supernatant layer.

FIG. 20 is a graphic representation of data showing the gross quantityof MSCs per 10 cc of bone marrow aspirate for MCSs obtained from thebuffy coat and the adipose supernatant layer.

FIG. 21 is a graphic representation of data showing the number of MSCsinitially obtained from 10 c.c. of bone marrow from both the buffy coatand lipid layer fractions.

FIG. 22 is a graphic representation of data showing the number of daysin culture required to obtain the quantity of adipose and buffy coatMSCs derived from 10 c.c. bone marrow necessary to passage.

FIG. 23 is a graphic representation of MSC population doubling times foradipose and buffy coat MSCs derived from 10 c.c. bone marrow.

FIG. 24 is a graphic representation of increased P₀MSC yield comparingemulsified and non-emulsified samples obtained from the same patient.

DETAILED DESCRIPTION

Unless otherwise indicated, all numbers expressing quantities ofingredients, dimensions reaction conditions and so forth used in thespecification and claims are to be understood as being modified in allinstances by the term “about”.

In this application and the claims, the use of the singular includes theplural unless specifically stated otherwise. In addition, use of “or”means “and/or” unless stated otherwise. Moreover, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one unit unless specifically statedotherwise.

Mesenchymal stem cells (MSCs) can be obtained from fractionated bonemarrow aspirate. Bone marrow aspirate may be fractionated using acentrifuge-based or similar technique which separates the aspiratedfluid into density graded layers. As shown in FIG. 1, fractionated bonemarrow aspirate 100 typically includes at least the following layers,ordered from greater to lesser density, a red blood cell (RBC) layer102, a buffy coat layer 104, a serum layer 106 and an adiposesupernatant layer 108. Conventional techniques for the extraction ofMSCs from bone marrow aspirates typically feature the isolation andprocessing of the buffy coat layer 104. Many different methods have beenused to isolate the buffy coat. Conventional buffy coat isolation andprocessing methods discard the adipose layer supernatant 108.

C. L. Insausti, M. B. Blanquer, L. M. Olmo, M. C. Lopez-Martinez, X. F.Ruiz, F. J. Lozano, V. C. Perianes, C. Funes, F. J. Nicolas, M. J.Majado, and J. M. Jimenez, ‘Isolation and Characterization ofMesenchymal Stem Cells from the Fat Layer on the Density GradientSeparated Bone Marrow’, Stem Cells Dev, 21 (2012), 260-72. (Insausti)first disclosed in 2012 that the adipose layer of fractionated bonemarrow aspirate contains MSCs. It was estimated by Insausti thatprocessing the adipose layer along with the buffy coat might increasestem cell yields from a bone marrow draw by as much as approximately50%. The methods and apparatus disclosed herein may be used to isolate,collect and process the adipose layer 108 of fractionated bone marrowaspirate, with or without co-processing of the buffy coat.Alternatively, the apparatus and methods disclosed herein may be used toobtain MSCs from other non-marrow sources of adipose tissue.Surprisingly and advantageously, applicants have demonstrated MSC yieldsfrom the adipose layer of bone marrow aspirate which are increased in anamount significantly greater than 50% when compared to the MSC yieldobtained when processing the buffy coat alone.

As noted above, Insausti estimated that processing the adipose layeralong with the buffy coat might increase stem cell yields from a bonemarrow draw by as much as approximately 50%. This relatively modestyield was in part caused by difficulty encountered in extracting theMSCs from the surrounding adipose tissue. In particular, applicantsbelieve that the MSCs in the adipose layer supernatant 108 offractionated marrow aspirate (or the MSC's in other adipose tissue) maybe locked in a fine collagen matrix. For example, abdominal subcutaneousfat has a strong collagen matrix that must be disrupted with chemicaldigestion before viable stem cells can be obtained. Applicants havedetermined that mechanical emulsification of the adipose fraction ofbone marrow aspirate can greatly increase the MSC yield to valuessignificantly above the 50% increase estimated by Insausti.

Specifically, as detailed below, the novel step of applying mechanicalemulsification to adipose layer supernatant resulted in an increased MSCyield by approximately 700%. Applicants believe that the increased MSCyield when compared to Insausti et. al. is due to the mechanicaldissociation of stem cells from the finer collagen matrix of thistissue.

Accordingly, the present disclosure provides device embodiments, systemsand methods for isolating the stem cell rich adipose layer supernatant108 (alternatively referred to herein as the adipose LS 108) of wholebone marrow aspirate. Embodiments may optionally include isolating andco-processing the buffy coat layer 104. Embodies may also be applied, incertain instances, to other sources of adipose tissue.

One family of system embodiments feature a closed system suitable foruse in a physician's office for the withdrawal of marrow from a patientfollowed by the substantially contemporaneous rapid isolation of theadipose LS 108 and re-injection or surgical placement of adipose LS 108or MSCs isolated therefrom into the patient to enhance tissue repair. Inanother family of embodiments the system may be open ended or partiallyopen ended such that adipose LS or MSCs isolated therefrom are expandedor otherwise processed before reintroduced into the patient to achievetherapeutic goals.

Device embodiments may be used to isolate adipose LS 108 alone or incombination with the buffy coat 104 of a whole marrow aspirate. Deviceembodiments may also combine the adipose LS 108 with one or morecomponents of the bone marrow aspirate such as the serum layer 106, anisolated fraction of the serum layer and/or buffy coat 104 and/or RBClayer 102 such as platelets or white blood cells.

Method embodiments may be performed manually or automatically orsemi-automatically with appropriate devices. Accordingly, certainautomated devices incorporate optical sensors or other detectors toidentify the various marrow fractions of interest such as the adipose LS108, serum 106, buffy coat 104, or RBC layer 102.

In one specific device embodiment, as shown in FIG. 2A, a centrifugetube 110 fabricated to have multiple chambers as described below iscentrifuged at 100-500 g for 5-10 minutes. The centrifuge tube 110 isprovided with a plurality of chambers. The first chamber 112 performs asa typical centrifuge chamber to produce the fractionated bone marrowaspirate with the adipose LS on top 108, the serum layer 106 below theadipose LS, the buffy coat 104 below the serum layer, and the RBC layer102 below the buffy coat. The centrifuge tube 110 also includes at leasta secondary chamber 114 and a tertiary chamber 116 which, for example,provide for the serum layer 106 to be decanted into the secondarychamber 114 and the adipose LS 108 to be collected into the tertiarychamber 116.

In use, the centrifuge tube 110 is centrifuged as described above toseparate the bone marrow aspirate into layers, as illustrated in FIG.2A. A second centrifuge run is performed to decant the serum layer 106into the secondary chamber 114 as illustrated in FIG. 2B. A thirdcentrifuge run performed in an upright position may then eject theadipose LS 108 and the buffy coat 104 into the tertiary chamber 116.

An alternative device embodiment is illustrated in FIGS. 3-4. In thisalternative embodiment, certain layers, for example the adipose LS 108,serum 106, and buffy coat 104 are collected in the secondary chamber 114of a dual chamber centrifuge tube 110 and then the adipose LS 108 (withor without the buffy coat) is isolated via the insertion of a tube,catheter, or needle 122, into the secondary chamber 114 such that theserum layer between the adipose LS and buffy coat is collected or drawninto a withdrawal chamber 124 which can be, but is not limited to aconventional syringe. The serum 106 can then be expelled from thewithdrawal chamber 124 and the same chamber 124 can be used to collectthe adipose LS 108 and/or buffy coat 104 or a fourth chamber can be usedfor adipose LS or buffy coat collection. As shown in FIG. 3A, 3B. 3C and4, a density-tuned floating element 126 may be used to cause the adiposeLS (or other layers, depending on the density of the density-tunedfloating element 126) to collect in the secondary chamber 114 forefficient withdrawal. The use of density-tuned floating elements 126 isdescribed in more detail below.

With respect to the embodiment of FIG. 3A, 3B, and 3C a first centrifugerun can be performed to fractionate the bone marrow aspirate asdescribed above. Then, as shown in FIG. 3A, a second centrifuge run(performed with or without the addition of a density tuned floatingelement 126 to the centrifuge tube 110) may separate the buffy coatlayer 104, serum layer 106, and adipose LS 108 in the secondary chamber114. As shown in FIG. 3B, the serum layer 106 positioned between theadipose LS 108 and buffy coat layer 104 may be withdrawn into thewithdrawal chamber 124. This step may be followed by withdraw of thevaluable adipose LS 108 and buffy coat layer 104 as shown in FIG. 3C.

In an alternative device embodiment, as shown in FIG. 5, after theinitial centrifuge run as described above, the centrifuge tube 128 isprovided with a novel cap 130 supporting a tube 132 inserted to thecorrect depth to collect the adipose LS 108 from the centrifuge tube128. Thus, the adipose LS 108 may be withdrawn into a withdraw chamber124. The withdraw chamber 124 may be a syringe, later used for directinjection of the adipose LS 108 for therapeutic purposes, connected tothe centrifuge chamber 128 by a Luer lock or otherwise firmly attachedto the tube 132. The depth of the tube 132 may, in certain embodiments,be manually or automatically adjusted to the correct depth to optimizeadipose LS 108 recovery.

In yet another device embodiment as shown in FIG. 6A, 6B, and 6C, acentrifuge tube 134 is provided with a plunger 136 (which may or may notbe detachable). As shown in FIGS. 6A-6C, the plunger equipped centrifugetube 134 may be used to expel the RBC fraction 102, the buffy coat 104,and serum 106 from the inferior end of the tube 134. This allows theadipose LS 108 to remain in the chamber 134. In certain embodiments thecentrifuge tube 134 may be implemented with a suitably sized syringethat provides for direct clinical injection of the isolated adipose LS108 into a patient with or without serum.

In the alternative embodiment of FIG. 7A and 7B, a first chamber 138 isprovided in the form of a disc that fractionates the whole marrow byspinning around a central axis. The adipose LS 108, being the leastdense fraction, collects in the middle of the disc and may then be drawnoff into a second, collection chamber 140 which may or may not be asyringe which would also provide for direct clinical use of the adiposeLS 108. In certain embodiments, the collection chamber 140 may beconnected to the first chamber 138 as shown in FIG. 7B. And opening orgate may be provided between the chambers 138, 140 such that the adiposeLS 108 may be forced, expelled or otherwise drawn into the chamber 140during the fractionation process. A similar embodiment, illustrated inFIG. 8A and 8B, includes a first vertical separation tube 142 thatrotates about its vertical axis causing a fractioning pattern asdescribed above with the additional creation of a fluid meniscus wherethe adipose LS 108 in isolated in the center of a depression. Theadipose LS may then be isolated and withdrawn using any of the methodsdescribed above.

FIGS. 9A, 9B, and 10 illustrate alternative types of centrifuge tubesuitable for use with certain embodiments described above. For examplethe centrifuge tube of FIG. 9A is implemented as a specialized tubularchamber 144 including a superior portion 146 which is restricted indiameter relative to the inferior portion 148. This configuration allowsthe superior adipose LS layer 108 to be elongated after fractionationfor easier manual or automatic removal. This system may be part of aclosed system providing for direct therapeutic use of the adipose LS108, once isolated or part of an open system where the adipose LS 108 isfor the process before therapeutic use.

In yet another centrifuge tube embodiment (FIG. 9B), a speciallyfabricated density-tuned floating disc 126 is provided having a selectedspecific gravity that causes the disc 126 to float just below theadipose LS 108 and above the serum 106 after fractionation, allowing foreasier manual or automatic removal of the adipose LS 108. In a similarembodiment illustrated in FIG. 10, a two-chambered centrifuge tube 110utilizes the specific gravity or density-tuned disc 126 to act as astopper that can be affixed to the side walls of the primary chamber 112of the centrifuge tube 110 after the disc 126 floats just above theserum layer 106 and below the adipose LS 108. The adipose LS 108 canthen be manually or automatically decanted into a secondary chamber 114for isolation.

In an alternative device embodiment illustrated in FIG. 11, thecentrifuge tube is implemented as a superiorly tapered tube 154 with aninferior plunger 156. The superiorly tapered tube 154 is attacheddirectly, or via a tube 158, to a withdraw chamber 124. The superiortapered portion 160 of the tube 154 provides for the adipose LS 108 tobe elongated and selectively pushed via the inferior plunger 156 ordrawn through suction into the withdraw chamber 124 for isolation. Eachtube or chamber 154, 124 may in certain embodiments be implemented as asyringe providing for the direct closed loop clinical use of the adiposeLS 108.

In an alternative device embodiment illustrated in FIG. 12, thecentrifuge tube 110 is implemented with a cap 130 on the superior endand a highly lipophilic porous membrane 168 positioned at or near thesuperior end of the 130. In use, a port and withdrawal tube 170 may beconnected to a withdraw chamber, possibly implemented as a syringe or avacuum line such that the adipose LS 108 may be lifted out of thefractionated solution opposite the porous highly lipophilic membrane168.

Optionally, as shown in FIG. 13 saline 172 or another biologically inertfluid may be added to the centrifuge tube 110 to float the adipose LS108 and lift the adipose LS 108 into contact with the lipophilicmembrane 168. Then, as shown in FIG. 14, the lipophilic membrane 168 canthen be washed to displace the adipose LS 108 into an empty tube 110where the adipose LS 108 may be collected by withdraw into a withdrawchamber 124 which may be implemented with a syringe providing for thedirect clinical use of the adipose LS 108.

In another device embodiment illustrated in FIG. 15, the adipose LS 108(which can optionally be intermixed slightly with the fibrinogen richserum 106) is polymerized via the addition of thrombin, CaCl₂, oranother clotting agent 174. Then the polymerized adipose LS 176 iseither manually or automatically removed from the top of the serum layeror drawn though a tube into a withdrawal chamber 124.

In yet another device embodiment illustrated in FIG. 16, the centrifugetube 110 is provided with a side port 178 or multiple side ports thatcan be attached to or be punctured by a needle 122 or other conduit thatconnects to a withdraw chamber 124 which may be implemented with asyringe. The side port 178 or side ports are located at or just belowthe adipose LS 108 boundary such that the adipose LS can be drawn intothe withdraw chamber 124 for isolation after fractionation.

In any of the above described embodiments the device may also contain anintegrated or separate well system that allows the isolated adipose LSto be processed such that the stem cells and other cellular componentsare separated from the fine collagen matrix present in the adiposetissue. Emulsification may be accomplished by mechanical or chemicalmeans. For example, as shown in FIG. 17A, an emulsification system 180may be provided as one or more additional chambers associated with thedevice or may be located in a separate apparatus such as a syringeproviding for clinical use with a patient. In one representative, butnon-limiting embodiment, the emulsification system 108 may use two ormore chambers or a single chamber to accomplish emulsificationprocessing. For example, emulsification may occur as the adipose LS 108is forced through a small aperture 182 between a first emulsificationchamber 184 and the second emulsification chamber 186. The adipose LS108 may be forced repeatedly through the aperture 182 to accomplish thedesired degree of emulsification.

Alternatively, as shown in FIGS. 17B and 17C a fine emulsification grate188 may be located in one chamber and physically passed through theisolated adipose LS 108 (FIG. 17B). Alternatively, the adipose LS tissuemay be passed from one chamber to another through an emulsificationgrate 188. Alternatively, the grate may be fixed with the adipose LS 108passing from one part of a single chamber to another part of the samechamber.

In alternative device embodiments, the adipose LS can be processed inany one of the above described chambers or an out board vessel with adigestion agent such as collagenase or lecithin to dissociate the cellsfrom the collagen matrix of the adipose LS 108. In other embodiments,the adipose LS can be processed using sonic energy or vibration todissociate the cellular components.

In other alternative device embodiments, the dissociated cells plus theremaining adipose LS structural tissue (collagen and oils) can befurther centrifuged to isolate a cell pellet that can then be washed.This pellet can then be added to the isolated bone marrow serum,platelets, RBCs, buffy coat, mesenchymal stem cells, other adult stemcells, or a nucleated cell mixture and/or isolated nucleated cell typesfor clinical use.

Alternative embodiments disclosed herein include methods of processingbone marrow aspirates and/or methods of collecting, preparing orreintroducing mesenchymal stem cells into an animal or human patient.Method embodiments include collecting bone marrow aspirate andfractionating the bone marrow aspirate to cause the formation of atleast an adipose layer supernatant 108. The adipose layer supernatantmay then be isolated utilizing one or more of the devices describedabove or similar devices suitable for isolating the adipose layersupernatant. For example, the bone marrow aspirate may be centrifuged tocause fractionation and the adipose layer supernatant withdrawn ordecanted according to the techniques described above, or other suitabletechniques.

The methods may further include processing the adipose layer to collectMSCs. For example, the adipose layer may be emulsified, mechanicallyemulsified, chemically digested, polymerized, subjected to sonic orvibrational energy, centrifuged or otherwise treated to aid with theextraction or collection of MSC's from the adipose layer tissue orfluid.

Upon collection, the adipose layer supernatant 108 or MSCs collectedtherefrom may be reintroduced into an animal or human patient to achievetherapeutic goals. In certain embodiments, bone marrow may be drawn; anadipose layer supernatant 108 collected and MSCs may be extractedtherefrom and reintroduced into the patient in a single closed-looptreatment session. Alternatively, MSCs or adipose layer supernatant maybe collected and stored or processed for subsequent use. For exampleMSCs collected and isolated as described herein may be expanded inculture prior to reintroduction into a patient for therapeutic purposes.

EXAMPLES

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention. As noted above,applicants have been able to collect surprisingly high quantities ofMSCs from bone marrow-derived adipose tissue when compared to thequantity of MSC's collected from similarly obtained buffy coat tissue.The results of preliminary laboratory investigations are described belowand graphically represented in FIGS. 18-23.

Example 1

10 cc of bone marrow aspirate was withdrawn from several patients.Following a brief centrifugation step of the whole bone marrow aspiratein a sterile conical tube at 200×g, the buoyant adipose layer wascollected manually via serological pipette along with a portion of bonemarrow aspirate serum. In an initial plating of this bone marrowfraction, a ‘dirty’ culture consisting of cell debris and ‘oily’substances in the native lipid layer was observed. These components weredifficult to remove in later media changes. Further, subsequentre-plating of the media containing lipid suspension resulted in theestablishment of large numbers of fibroblast-like morphologies in cellsbelieved to be MSCs. This indicated that the initial plating wassub-optimal and potentially resulted in discarding target cells, if notre-plated, thereby consuming additional resources and time.

Example 2

10 cc of bone marrow aspirate was withdrawn from seven patients. Theadipose-plasma solution was passed through a small gauge emulsifierseveral times to dissociate adipose cells from the associated MSCs. Thispreparation was used for cell counting, flow cytometric analysis and invitro plating for cell expansion.

Emulsification was employed in an effort to distort the lipid layermatrix to increase initial plating efficiency. Emulsification andplating resulted in an apparent increase of adherent cells compared tothose not emulsified derived from the same lipid sample (see Example 1).In addition, re-plating of the supernatant following 2 days in culturedid not result in the establishment of cells of the appropriatemorphology and the initial culture was easily cleaned of the featuresdescribed in the native layer. Therefore, mechanical disruption of thelipid layer via emulsification is believed to be optimal for initial invitro plating of the lipid layer, potentially by exposing suspected MSCsto the environment and allowing for adhesion.

A very significant difference in the number and percentage of cells thatstained positive for the stem cell markers CD44, CD73, CD90 and CD105was observed when comparing isolations from the buffy coat with theadipose layer. For example, FIG. 18 illustrates the results of a manualcell count for 7 patients and indicates a lower mono-nucleated cellcount (MNC) in the adipose layer (graph bars 200) compared to the buffycoat layer (graph bars 202). However, when compared to the contaminatingcell background, as shown in FIG. 19, the adipose layer afteremulsification demonstrated MSCs to comprise approximately 5% of thetotal cell population (graph bars 204). On the contrary, the buffy coatincludes only 0.01-0.001% MSCs as determined by flow cytometry analysis(graph bars 206).

Further, as shown in FIG. 20, the gross MSC count obtained from thebuffy coat (graph bars 208) ranged from approximately 50-300 cells per10 cc of bone marrow; while the gross MSC count obtained from theadipose layer collected and processed as described above (graph bars210) was determined to range from approximately 500-4000 cells per 10 ccof bone marrow.

Accordingly, the number of non-MSC ‘contaminating cells’ in the buffycoat layer of bone marrow is significant higher than in the adiposelayer; the percentage of MSCs in the buffy coat typically ranges from0.01-0.001% as compared to the adipose layer where the range appears tobe between 3%-15%. Based upon the data represented in FIGS. 19 and 20,it is believed that the number of MSCs in the adipose layer far exceedsthat of the buffy coat layer due to the large difference in thepercentage of MSCs that exist in the respective regions.

As shown in FIGS. 21-23, in vitro studies confirmed that elevated levelsof MSCs can be obtained from the bone marrow adipose layer afterprocessing bone marrow aspirate with the devices and methods describedherein versus comparable levels of buffy coat derived MSCs. FIG. 21depicts the number of MSCs expanded ex vivo from 10 cc. of bone marrowderived adipose tissue (graph bars 212) versus the number of MSCsexpanded ex vivo from 10 cc. of bone marrow derived buffy coat layer(graph bars 214). This data supports the foregoing flow cytometric dataof FIGS. 19 and 20) and it is clear that a significantly larger numberof MSCs were expanded from the adipose layer compared to the buffy coatlayer using the methods described.

As shown in FIG. 23, additional passages revealed that adipose derivedMSCs (graph bars 222) are also characterized by a lower doubling timeresulting from increased rate of division when compared to huffy coatderived MSCs seeded at the same cell density (graph bars 220). Thisindicates that the innate rate of division differs between the adiposederived and huffy coat derived MSCs, suggesting therapeutic advantagesfrom the adipose derived MSCs.

As shown in FIG. 23, additional passages revealed that adipose derivedMSCs (graph bars 220) are also characterized by a lower doubling timeresulting from increased rate of division when compared to buffy coatderived MSCs seeded at the same cell density (graph bars 222). Thisindicates that the innate rate of division differs between the adiposederived and buffy coat derived MSCs, suggesting therapeutic advantagesfrom the adipose derived MSCs.

Example 3

Bone marrow aspirate samples were withdrawn from three patients anddivided into equal volume subsamples to investigate the effect ofemulsification. One subsample from each patient was emulsified asdescribed herein. A 2nd subsample was not emulsified. The cells wereplated in a T-25 flask and grown in a 10% FBS/90% DMEM growth medium for6 days. As shown in FIGS. 24-25, for each patient, the cells from theemulsified culture (graph bars 224) initiate and proliferate faster whencompared to cells grown from the non-emulsified culture. In addition,the cell cultures prepared from emulsified samples were observed to becleaner and more easily cleared of debris with passage. Thus, thenon-emulsified samples appeared “dirty” for longer periods of time.

Various embodiments of the disclosure could also include permutations ofthe various elements recited in the claims as if each dependent claimwas a multiple dependent claim incorporating the limitations of each ofthe preceding dependent claims as well as the independent claims. Suchpermutations are expressly within the scope of this disclosure.

The description of the various embodiments has been presented forpurposes of illustration and description, but is not intended to beexhaustive or limiting of the embodiments to the fom1 disclosed. Thescope of the present disclosure is limited only by the scope of thefollowing claims. Many modifications and variations will be apparent tothose of ordinary skill in the art. The embodiments described and shownin the figures were chosen and described in order to best explain theprinciples of the disclosed embodiments, the practical application, andto enable others of ordinary skill in the art to understand the variousembodiments with various modifications as are suited to the particularuse contemplated.

The description of the various embodiments has been presented forpurposes of illustration and description, but is not intended to beexhaustive or limiting of the embodiments to the form disclosed. Thescope of the present disclosure is limited only by the scope of thefollowing claims. Many modifications and variations will be apparent tothose of ordinary skill in the art. The embodiments described and shownin the figures was chosen and described in order to best explain theprinciples of the disclosed embodiments, the practical application, andto enable others of ordinary skill in the art to understand the variousembodiments with various modifications as are suited to the particularuse contemplated.

What is claimed is:
 1. A method of processing bone marrow aspiratecomprising: providing a device for processing bone marrow aspiratecomprising: a first chamber; a second chamber in fluid communicationwith the first chamber; and a mechanical emulsification system in fluidcommunication with the second chamber; fractionating bone marrowaspirate within the first chamber of the device into layers including anadipose layer supernatant; collecting the adipose layer supernatant fromthe processed bone marrow aspirate in the second chamber; andemulsifying the adipose layer supernatant in the emulsification system.2. The method of claim 1 further comprising processing the adipose layersupernatant to collect mesenchymal stem cells.
 3. The method of claim 1further comprising adding a secondary substance to the bone marrowaspirate.
 4. The method of claim 3 wherein the secondary substancecomprises at least one of a biologically inert fluid, CaCl₂, thrombin, aclotting agent or a polymerization agent.
 5. The method of claim 1further comprising adding a digestion agent to the collected adiposelayer supernatant.
 6. The method of claims 5 wherein the digestion agentcomprises at least one of collagenase or lecithin.
 7. The method ofclaim 1 further comprising applying at least one of sonic energy orvibration to the adipose layer supernatant.
 8. The method of claim 1wherein the second chamber comprises at least one of a syringe, pipetteor tube in fluid communication with the adipose layer supernatant. 9.The method of claim 1 wherein the first chamber comprises one or moreof: a cap comprising a fluid access port providing for the secondchamber to be placed into fluid communication with the adipose layersupernatant; a plunger providing for the expulsion of a selected portionof the fractionated bone marrow aspirate from the first chamber; a diskshaped volume which provides for the collection of an adipose layersupernatant fraction at a central region of the disk shaped volume uponthe rotation of the first chamber around a central axis; a portion ofrestricted diameter positioned to correspond with the location of anadipose layer supernatant fraction upon fractionation of bone marrowaspirate placed within the first chamber; a floating disk having adensity selected to cause the disk to float substantially between aserum layer and an adipose layer supernatant fraction upon fractionationof the bone marrow aspirate; a porous lipophilic membrane providing forthe separation of an adipose layer supernatant fraction uponfractionation of the bone marrow aspirate; and one or more ports influid communication with an adipose layer supernatant fraction uponfractionation of the bone marrow aspirate.
 10. The method of claim 1wherein the mechanical emulsification system comprises a firstemulsification chamber and a second emulsification chamber in fluidcommunication with each other through an aperture sized to provideemulsification upon passage of the adipose layer supernatant between thefirst and second emulsification chambers.
 11. The method of claim 1wherein the mechanical emulsification system comprises a firstemulsification chamber and a second emulsification chamber in fluidcommunication with each other through an emulsification screen providingfor the emulsification of the adipose layer supernatant upon passage ofadipose layer supernatant between the first and second emulsificationchambers.
 12. The method of claim 1 wherein the mechanicalemulsification system comprises an emulsification screen movable withrespect to the adipose layer and providing for the emulsification of theadipose layer supernatant.